1. Field of the Invention
This invention generally relates to the detection of a polymorphic locus nucleotide sequence which can be used to identify an individual of a species.
2. Related Art
Mammalian genomes consist of unique DNA sequences interspersed with moderately and highly repetitive DNA sequences. Gene mapping by meiotic linkage analysis has traditionally been carried out using variations in unique sequence DNA, such as restriction fragment length polymorphisms (Botstein, et al., Am J. Hum. Genet, 32:314-331, 1980), as genetic markers. Recently, variations in the repetitive sequence elements such as minisatellite or variable number tandem repeat (VNTR) sequences (Jeffreys, et al., Nature, 314:67-73, 1985; Nakamura, et al., Science, 235:1616-1622, 1987), and microsatellite or variable simple sequence motifs (VSSM) (Litt and Luty, Am. J. Hum. Genet., 44:397-401, 1989; Weber and May, Am. J. Hum. Genet., 44:388-396, 1989) have been found to be useful for linkage studies. One advantage to the use of repetitive sequence variations rather than unique sequence variations is the apparent greater number of alleles present in normal populations when compared to restriction fragment length polymorphisms (RFLPs). A second advantage is the ability to readily detect sequence length variations using the polymerase chain reaction to facilitate the rapid and inexpensive analysis of large numbers of DNA samples.
Microsatellite elements consist of simple mon-, di-, or tri-nucleotide sequences where alleles differ by one or more repeat units (Luty, et al, Am.J. Hum. Genet., 46:776-783, 1990; Tautz, et al., Nature, 322:652-656, 1986; Weber and May, Am. J. Hum. Genet., 44:388-396, 1989). Minisatellites, or VNTR sequences, typically have a repeat unit of 20 to several hundred nucleotides and alleles differ by as little as one repeat unit. Among simple sequences, the (TG).sub.n or (CA).sub.n repeat elements have recently proven extremely useful for meiotic mapping since (1) they are abundant in the genome, (2) display a large number of different alleles, and (3) can be rapidly assayed using the polymerase chain reaction (Litt and Luty, Am. J. Hum. Genet., 44:397-401, 1989; Weber and May, Am. J. Hum. Genet, 44:388-396, 1989).
A number of other short sequence motifs have been found in mammalian (Hellman, et al., Gene, 68:93-100, 1988; Knott, et al., Nuc. Acids Res., 14:9215-9216, 1986; Litt and Luty, Am. J. Hum. Genet, 44:397-401, 1989; Milstein, et al., Nuc. Acids Res., 12:6523-6535, 1984; Stoker, et al., Nuc. Acids Res., 13:4613-4621, 1985; Vassart, et al., Science, 233:683-684, 1987; and Vergnaud, Nuc. Acids Res., 17:7623-7630,1989), and avian (Gyllensten, et al., Nuc. Acids Res., 17:2203-2214, 1989; Longmire, et al., Genomics, 2:14-24, 1988) genomes and are thought to accumulate by DNA slippage during replication (Tautz, et al., Nature, 322:652-656, 1986) or unequal recombination events (Wolff, et al., Genomics, 5:382-384, 1989). Many of these repeat elements display a high degree of genetic variation and thus are also useful for meiotic mapping.
The VNTR sequence isolated by Jeffreys contains an invariant core sequence GGGCAGGAXG (SEQ ID NO:1) which bears some similarities to the chi sequence of phage lambda (Wolff, et al., Genomics, 5:382-384, 1989) and is detected by a restriction fragment of bacteriophage M13 (Vassart, et al., Science, 233:683-684, 1987). Similar repeat elements have been detected by Nakamura, et al. (Science, 235:1616-1622, 1987) and contain a similar, but distinctive, common core unit GGG--GTGGGG (SEQ ID NO:2). Elements of this type occur within several known gene sequences including the .beta. globin locus. Similar VNTR elements have been described within the apolipoprotein B (Boerwinkle, et al., Proc. Natl. Acad. Sci. USA, 86:212-216, 1989; Knott, et al., Nuc. Acids Res., 14:9215-9216, 1986) and collagen type II genes (Stoker, et al., Nuc. Acids Res., 13:4613-4621, 1985) and contain a distinct AT-rich motif. Though a physiological function for repetitive elements of this type has not been defined, they have been suggested as potential hot spots for chromosome recombination (DeBustros, et al., Proc. Natl. Acad. Sci., 85:5693-5697, 1988) or elements important for the control of gene expression (Hellman, et al., Gene, 68:93-100, 1988; Milstein, et al., Nuc. Acids Res., 12:6523-6535, 1984).
In addition to linkage analysis, highly variable probes are useful for identity testing in practical applications such as paternity testing and forensics. One disadvantage to the use of VNTR probes, is the necessity for carrying out Southern blot analysis for allele detection. Frequently DNA samples for these applications are of poor quality and low quantity. The analysis of (CA).sub.n polymorphisms may be difficult since the elements may be short and alleles may differ by as little as 2 nucleotides, necessitating the use of DNA sequencing gels and radioactive labeling for allele detection (Litt and Luty, Am. J. Hum. Genet., 44:397-401, 1989; Weber and May, Am. J. Hum. Genet., 44:388-396, 1989). Consequently, an unfulfilled need exists for a technique which can detect a unique DNA sequence in specimens of poor quality or in low quantity without having to rely on isotopic detection. The present invention provides such a technique.